LINC00662通过miR-144/COX-2调控胶质瘤细胞替莫唑胺耐药性的作用研究

Mechanism of LINC00662 regulating temozolomide resistance of glioma cells via miR-144/COX-2 signaling

目的探究LINC00662通过miR-144/COX-2轴调节胶质瘤细胞耐药性的机制。方法qRT-PCR检测胶质瘤组织和癌旁正常组织、U251细胞和U251/替莫唑胺(TMZ)细胞中LINC00662、miR-144和COX-2 mRNA表达水平;双荧光素酶报告系统评估LINC00662、miR-144和COX-2的调控关系。设置空白对照组、敲低LINC00662阴性对照(si-NC)组、敲低LINC00662组、同时敲低LINC00662和抑制miR-144表达组。CCK-8和Edu法分析细胞增殖情况;流式细胞术定量检测细胞凋亡;Western blot分析靶蛋白表达水平。结果与癌旁正常组织相比,胶质瘤组织中LINC00662和COX-2 mRNA表达显著上调,miR-144表达下调(P<0.05);与U251细胞相比,U251/TMZ细胞中LINC00662和COX-2 mRNA表达显著上调,miR-144表达下调(P<0.05)。双荧光素酶报告实验证实LINC00662靶向结合miR-144,miR-144靶向结合COX-2。与si-NC组相比,敲低LINC00662组U251/TMZ细胞增殖能力显著降低,凋亡率升高(P<0.05);COX-2、PCNA、MRP1和Bcl-2蛋白显著下调,Bax蛋白上调(P<0.05);抑制miR-144表达可在一定程度上逆转LINC00662敲低对U251/TMZ细胞增殖、凋亡的影响(P<0.05)。结论LINC00662通过miR-144/COX-2信号可调节胶质瘤细胞增殖、凋亡,改变胶质瘤细胞TMZ耐药性。

Objective To explore the mechanism of LINC00662 in regulation of temozolomide resistance of glioma cells through miR-144/COX-2 signaling.Methods qRT-PCR were used to measure the mRNA expression levels of LINC00662,miR-144,and COX-2 in glioma tissues,normal tissue adjacent to cancer,U251 cells,and U251/temozolomide(TMZ)cells.The dual-luciferase reporter system were used to assess the regulatory relationship of LINC00662,miR-144,and COX-2.The five groups were a blank control,knockdown LINC00662 negative control(si-NC),knockdown LINC00662,and simultaneous knockdown of LINC00662 and inhibition of miR-144 expression.Cell proliferation were analyzed by CCK-8 and Edu assays.Apoptosis was evaluated by flow cytometry.Expression levels of target proteins were analyzed by Western blot.Results Compared with adjacent normal tissue,the mRNA expression levels of LINC00662 and COX-2 were significantly upregulated and the expression level of miR-144 was downregulated in glioma tissues(P<0.05).Compared with U251 cells,the mRNA expression levels of LINC00662 and COX-2 were significantly upregulated and the expression level of miR-144 was downregulated in U251/TMZ cells.Dual-luciferase reporter assays showed that LINC00662 targeted miR-144,and miR-144 targeted COX-2.Compared with the si-NC group,cell proliferation of the knockdown LINC00662 group was significantly decreased,the apoptosis rate of the knockdown LINC00662 group was increased(P<0.05),COX-2,PCNA,MRP1 and Bcl-2 protein expression levels in the knockdown LINC00662 group were significantly downregulated,and the Bax protein expression level in the knockdown LINC00662 group was upregulated(P<0.05).Inhibiting miR-144 expression reversed the effects of LINC00662 knockdown on U251/TMZ cell proliferation and apoptosis(P<0.05).Conclusions LINC00662 regulates the proliferation and apoptosis of glioma cells through miR-144/COX-2 signaling and is closely related to temozolomide resistance of glioblastoma cells.